Zebrafish live imaging reveals a surprisingly small percentage of spinal cord motor neurons die during early development

  1. Hao Jia
  2. Hongmei Yang
  3. Kathy Qian Luo

  • Curated by eLife

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    eLife Assessment

    The authors have developed a biosensor for programmed cell death. They use this biosensor to provide cell death measurements in a specific early development time. The findings useful in a specific context; however, the application of this biosensor is incomplete as it does not take into account existing literature and is missing controls.

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Abstract

It is widely accepted that large numbers of neurons die during the early development of vertebrates; however, the tracking of this dying process in live animals remains challenging. Here, we generated sensor zebrafish achieving live imaging of motor neuron apoptosis at single- cell resolution. Using these sensor zebrafish, we observed for the first time that in an apoptotic motor neuron, caspase-3 activation occurred quickly within 5-6 min and at the same time between the cell body and axon. Interestingly, we found that only a surprisingly small percentage of spinal cord motor neurons died during zebrafish early development, which is quite different from the generally believed massive motor neuron death occurred in the embryonic stage of chicks, mice, rats, and humans. We also observed that most of the apoptotic bodies of dead motor neurons were not colocalized with macrophages. These sensor zebrafish can serve as powerful tools to study motor neuron apoptosis in vivo .

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  1. Version published to 10.7554/elife.95691.3 on eLife
  2. Version published to 10.7554/elife.95691 on eLife
  3. eLife

    eLife Assessment

    The authors have developed a biosensor for programmed cell death. They use this biosensor to provide cell death measurements in a specific early development time. The findings useful in a specific context; however, the application of this biosensor is incomplete as it does not take into account existing literature and is missing controls.

  4. eLife

    Joint Public Review:

    Summary:

    Jia and colleagues developed a fluorescence resonance energy transfer (FRET)-based biosensor to study programmed cell death in the zebrafish spinal cord. They applied this tool to study death of zebrafish spinal motor neurons.

    Strengths:

    Their analysis shows that the tool is a useful biosensor of motor neuron apoptosis in living zebrafish and can reveal which part of the neuron undergoes caspase activation first.

    Weaknesses:

    As far as it is possible to tell, the authors focus on death of motor neurons innervating axial muscles. Previous work from over 30 years ago revealed that only a small number of these motor neurons die early in development. So this is not new, although following the cells and learning details of their apoptosis is new. Most of the work on motor neuron death in tetrapods was carried out on limb innervating motor neurons. Zebrafish have paired pectoral and pelvic fins, homologs of tetrapod paired limbs. These fins are innervated by distinct sets of motor neurons in zebrafish, as they are in tetrapods. However, the authors have not focused on these particular motor neurons, and thus have not made a fair comparison with tetrapods. In fact, they do not tell us which spinal levels they observed or whether they have been consistent from animal to animal. Pelvic fins emerge much later than pectoral fins in zebrafish, so it is possible that the time frame during which the authors imaged motor neuron death does not include motor neurons innervating pelvic fins.

  5. eLife

    Author response:

    The following is the authors’ response to the previous reviews.

    Reviewer 1:

    (1) The results do not support the conclusions. The main "selling point" as summarized in the title is that the apoptotic rate of zebrafish motorneurons during development is strikingly low (~2% ) as compared to the much higher estimate (~50%) by previous studies in other systems. The results used to support the conclusion are that only a small percentage (under 2%) of apoptotic cells were found over a large population at a variety of stages 24-120hpf. This is fundamentally flawed logic, as a short-time window measure of percentage cannot represent the percentage on the long-term. For example, at any year under 1% of human population die, but over 100 years >99% of the starting group will have died. To find the real percentage of motorneurons that died, the motorneurons born at different times must be tracked over long term, or the new motorneuron birth rate must be estimated. Similar argument can be applied to the macrophage results.

    In the revised manuscript (revised Figure 4), we extended the observation time window as long as possible, from 24 hpf to 240 hpf. After 240 hpf, the transparency of zebrafish body decreased dramatically, which made optical imaging quite difficult.

    We are confident that this 24-240 hpf time window covers the major time window during which motor neurons undergo programmed cell death during zebrafish early development. We chose the observation time window based on the following two reasons: 1) Previous studies showed that although the time windows of motor neuron death vary in chick (E5-E10), mouse (E11.5-E15.5), rat (E15-E18), and human (11-25 weeks of gestation), the common feature of these time windows is that they are all the developmental periods when motor neurons contact with muscle cells. The contact between zebrafish motor neurons and muscle cells occurs before 72 hpf, which is included in our observation time window. 2) Most organs of zebrafish form before 48-72 hpf, and they complete hatching during 48-72 hpf. Food-seeking and active avoidance behaviors also start at 72 hpf, indicating that motor neurons are fully functional at 72 hpf.

    Previous studies in zebrafish have shown that the production of spinal cord motor neurons largely ceases before 48 hpf, and then the motor neurons remain largely constant until adulthood (doi: 10.1016/j.celrep.2015.09.050; 10.1016/j.devcel.2013.04.012; 10.1007/BF00304606; 10.3389/fcell.2021.640414). Our observation time window covers the major motor neuron production process. Therefore, we believe that neurogenesis will not affect our findings and conclusions.

    Although we are confident that 240 h tracking is long enough to measure the motor neuron death rate, several sentences have been added in the discussion part, “In our manuscript, we tracked the motor neuron death in live zebrafish until 240 hpf, which was the longest time window we could achieve. But there was still a possibility that zebrafish motor neurons might die after 240 hpf.”

    We agreed that the “2%” description might not be very accurate. Thus, we have revised our title to “Zebrafish live imaging reveals a surprisingly small percentage of spinal cord motor neurons die during early development.”

    (2) The conclusion regarding timing of axon and cell body caspase activation and apoptosis timing also has clear issues. The ~minutes measurement are too long as compared to the transport/diffusion timescale between the cell body and the axon, caspase activity could have been activated in the cell body and either caspase or the cleaved sensor move to the axon in several seconds. The authors' results are not high frequency enough to resolve these dynamics. Many statements suggest oversight of literature, for example, in abstract "however, there is still no real-time observation showing this dying process in live animals.".

    Real-time imaging of live animals is quite challenging in the field. Currently, using confocal microscopy, we can only achieve minute-scale tracking. In the future, with more advanced imaging techniques, the sensor fish in the present study may provide us with more detailed information on motor neuron death. We have removed “real-time” from our revised manuscript. We also revised the mentioned sentence in the abstract.

    (3) Many statements should use more scholarly terms and descriptions from the spinal cord or motorneuron, neuromuscular development fields, such as line 87 "their axons converged into one bundle to extend into individual somite, which serves as a functional unit for the development and contraction of muscle cells"

    We have removed this sentence.

    (4) The transgenic line is perhaps the most meaningful contribution to the field as the work stands. However, mnx1 promoter is well known for its non-specific activation - while the images do suggest the authors' line is good, motorneuron markers should be used to validate the line. This is especially important for assessing this population later as mnx1 may be turned off in mature neurons. The author's response regarding mnx1 specificity does not mitigate the original concern.

    The mnx1 promoter has been widely used to label motor neurons in transgenic zebrafish. Previous studies have shown that most of the cells labeled in the mnx1 transgenic zebrafish are motor neurons. In this study, we observed that the neuronal cells in our sensor zebrafish formed green cell bodies inside of the spinal cord and extended to the muscle region, which is an important morphological feature of the motor neurons.

    Furthermore, a few of those green cell bodies turned into blue apoptotic bodies inside the spinal cord and changed to blue axons in the muscle regions at the same time, which strongly suggests that those apoptotic neurons are not interneurons.

    In fact, no matter what method is used, such as using antibodies to stain specific markers to label motor neurons, 100% specificity cannot be achieved. More importantly, although the mnx1 promoter might have labeled some interneurons, this will not affect our major finding that only a small percentage of spinal cord motor neurons die during the early development of zebrafish.

    Reviewer 2:

    (1) Title: The 50% figure of motor neurons dying through apoptosis during early vertebrate development is not precisely accurate. In papers referenced by the authors, there is a wide distribution of percentages of motor neurons that die depending on the species and the spinal cord region. In addition, the authors did not examine limb-innervating motor neurons, which are the ones best studied in motor neuron programmed cell death in other species. Thus, a better title that reflects what they actually show would be something like "A surprisingly small percentage of early developing zebrafish motor neurons die through apoptosis in non-limb innervating regions of the spinal cord."

    In fish, there are no such structures as limbs, although fins may be evolutionarily related to limbs. In our manuscript, we studied the naturally occurring motor neuron death in the whole spinal cord during the early stage of zebrafish development. The death of motor neurons in limb-innervating motor neurons has been extensively studied in chicks and rodents, as it is easy to undergo operations such as amputation. However, previous studies have shown this dramatic motor neuron death occurs not only in limb-innervating motor neurons but also in other spinal cord motor neurons (doi: 10.1006/dbio.1999.9413).

    We have revised our title to “Zebrafish live imaging reveals a surprisingly small percentage of spinal cord motor neurons die during early development.”

    (2) lines 18-19: "embryonic stage of vertebrates" is very broad, since zebrafish are also vertebrates; it would be better to be more specific

    lines 25-26: The authors should be more specific about which animals have widespread neuronal cell death.

    We have revised our manuscript accordingly.

    (3) lines 98-99; 110-111; 113; 122-123; 140-141: A cell can undergo apoptosis. But an axon, which is only part of a cell, cannot undergo apoptosis. Especially since the axon doesn't have a separate nucleus, and the definition of apoptosis usually includes nuclear fragmentation. A better subheading would describe the result, which is that caspase activation is seen in both the cell body and the axon.

    We have revised the subheadings and related words in the manuscript accordingly. In the introduction, we also revised the expression of the third aim from “Which part of a neuron (cell body vs. axon) will die first?” to “Which part of a neuron (cell body vs. axon) will degrade first?”.

    (4) lines 159-160; 178-179: This is an oversimplification of the literature. The authors should spell out which populations of motor neuron have been examined and say something about the similarities and difference in motor neuron death.

    We have revised it accordingly.

    (5) lines 200; 216: The authors did not observe macrophages engulfing motor neurons. But that does not mean that they cannot. Making the conclusion stated in this subheading would require some kind of experiment, not just observations.

    We did observe few colocalizations of macrophages and dead motor neurons. To more accurately express these data, in the revised manuscript, we used “colocalization” to replace “engulfment.” The subheading has been revised to “Most dead motor neurons were not colocalized with macrophages.” Accordingly, panel C of Figure 5 has also been revised.

    (6) lines 234-246: The authors seem to have missed the point about VaP motor neuron death, which was two-fold. First, VaP death has been previously described, thus it could serve as a control for the work in this paper, especially since the conditions underlying VaP death and survival have been experimentally tested. Second, they should acknowledge that previous work showed that at least some motor neuron death in zebrafish differs from that described in chick and rodents. This conclusion came from work showing that death of VaP is independent of limitations in muscle innervation area, suggesting it is not coupled to muscle-derived neurotrophic factors.

    Figures: The authors should say which level of the spinal cord they examined in each figure.

    We have compared our findings with previous findings in the revised manuscript. The death of VaP motor neurons is not related to neurotrophic factors, but the death of other motor neurons may be related to neurotrophic factors, which needs further study and evidence. Our study examined the overall motor neuron apoptosis regardless of the causes and locations. To avoid misunderstanding, in the revised manuscript, we removed the data and words related to neurotrophic factors.

    We also extended the observation time window as long as possible, from 24 hpf to 240 hpf (revised Figure 4). After 240 hpf, the transparency of zebrafish body decreased dramatically, which made the optical imaging quite difficult.

  6. Version published to 10.1101/2024.01.26.577434v3 on bioRxiv
  7. eLife

    eLife Assessment

    This study estimates the fraction of apoptotic motor neurons during the development of the zebrafish spinal cord. The results are useful, but incomplete. Importantly, the data are inadequate to support the title or the conclusions presented in the abstract. A correct title could be: "A surprisingly small percentage of early developing zebrafish motor neurons die through apoptosis in non-limb innervating regions of the spinal cord."

  8. eLife

    Reviewer #1 (Public review):

    Summary:

    The authors aim at measuring the apoptotic fraction of motorneurons in developing zebrafish spinal cord to assess the extent of neuronal apoptosis during the development of of a vertebrate embryo in an in vivo context

    Strengths:

    The transgenic fish line tg(mnx1:sensor C3) appears to be a good reagent for motorneuron apoptosis studies, while further validation of its motorneuron specificity should be performed

    Weaknesses:

    The results do not support the conclusions. The main "selling point" as summarized in the title is that the apoptotic rate of zebrafish motorneurons during development is strikingly low (~2% ) as compared to the much higher estimate (~50%) by previous studies in other systems. The results used to support the conclusion are that only a small percentage (under 2%) of apoptotic cells were found over a large population at a variety of stages 24-120hpf. This is fundamentally flawed logic, as a short-time window measure of percentage cannot represent the percentage on the long-term. For example, at any year under 1% of human population die, but over 100 years >99% of the starting group will have died. To find the real percentage of motorneurons that died, the motorneurons born at different times must be tracked over long term, or the new motorneuron birth rate must be estimated.

    Similar argument can be applied to the macrophage results.

    The conclusion regarding timing of axon and cell body caspase activation and apoptosis timing also has clear issues. The ~minutes measurement are too long as compared to the transport/diffusion timescale between the cell body and the axon, caspase activity could have been activated in the cell body and either caspase or the cleaved sensor move to the axon in several seconds. The authors' results are not high frequency enough to resolve these dynamics

    Many statements suggest oversight of literature, for example, in abstract "however, there is still no real-time observation showing this dying process in live animals.".

    Many statements should use more scholarly terms and descriptions from the spinal cord or motorneuron, neuromuscular development fields, such as line 87 "their axons converged into one bundle to extend into individual somite, which serves as a functional unit for the development and contraction of muscle cells"

    The transgenic line is perhaps the most meaningful contribution to the field as the work stands. However, mnx1 promoter is well known for its non-specific activation - while the images do suggest the authors' line is good, motorneuron markers should be used to validate the line. This is especially important for assessing this population later as mnx1 may be turned off in mature neurons. The author's response regarding mnx1 specificity does not mitigate the original concern.

    Overall, this work does not substantiate its biological conclusions and therefore do not advance the field. The transgenic line has the potential for addressing the questions raised but requires different sets of experiments. The line and the data as reported are useful on their own by providing a short-term rate of apoptosis of the motorneuron population.

  9. eLife

    Reviewer #2 (Public review):

    Summary:

    Jia and colleagues developed a fluorescence resonance energy transfer (FRET)-based biosensor to study programmed cell death in the zebrafish spinal cord. They applied this tool to study death of zebrafish spinal motor neurons.

    Strengths:

    Their analysis shows that the tool is a useful biosensor of motor neuron apoptosis in living zebrafish and can reveal which part of the neuron undergoes caspase activation first, achieving two of their aims.

    Weaknesses:

    The third aim, to provide novel insights into the spatiotemporal properties and occurrence rates of motor neuron death requires additional context and investigation, especially to understand the significance of the differences they report between zebrafish motor neuron programmed cell death and what has been previously described in chicks and rodents. For example, mnx1 expresses not only in motor neurons, but also in interneurons. However, the way the authors counted living and dead cells does not take this into consideration, potentially underestimating the percentage of motor neurons that died. Previous studies of chicks and rodents showed widespread differences in the timing of motor neuron programmed cell death and the number of cells that died depending on the spinal cord region examined. The authors have not described which spinal cord segments they examined or whether they examined motor neurons in limb-bearing segments which have been best studied in other species. Previous literature investigated the death of an identified zebrafish motor neuron and provided experimental evidence that it is independent of limitations in muscle innervation area, suggesting it is not coupled to muscle-derived neurotrophic factors. Thus, the authors need to acknowledge that even previous to their study, there was literature suggesting that programmed cell death of at least one motor neuron in zebrafish does not easily fit into the "neurotrophic hypothesis" as it is generally formulated. Finally, the authors need to be mindful that showing that something does not happen in an observational study cannot reveal the capabilities of the cells involved without an experimental test.

  10. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Reviewer 1:

    We thank the reviewer for the time and effort in providing very useful comments and suggestions for our manuscript.

    (1) The results do not support the conclusions. The main "selling point" as summarized in the title is that the apoptotic rate of zebrafish motorneurons during development is strikingly low (~2% ) as compared to the much higher estimate (~50%) by previous studies in other systems. The results used to support the conclusion are that only a small percentage (under 2%) of apoptotic cells were found over a large population at a variety of stages 24-120hpf. This is fundamentally flawed logic, as a short-time window measure of percentage cannot represent the percentage in the long term. For example, at any year under 1% of the human population dies, but over 100 years >99% of the starting group will have died. To find the real percentage of motorneurons that died, the motorneurons born at different times must be tracked over the long term or the new motorneuron birth rate must be estimated. A similar argument can be applied to the macrophage results. Here the authors probably want to discuss well-established mechanisms of apoptotic neuron clearance such as by glia and microglia cells.

    We chose the time window of 24-120 hpf based on the following two reasons: 1) Previous studies showed that although the time windows of motor neuron death vary in chick (E5-E10), mouse (E11.5-E15.5), rat (E15-E18), and human (11-25 weeks of gestation), the common feature of these time windows is that they are all the developmental periods when motor neurons contact with muscle cells. The contact between zebrafish motor neurons and muscle cells occurs before 72 hpf, which is included in our observation time window of 24-120 hpf. 2) Zebrafish complete hatching during 48-72 hpf, and most organs form before 72 hpf. More importantly, zebrafish start swimming around 72 hpf, indicating that motor neurons are fully functional at 72 hpf. Thus, we are confident that this 24-120 hpf time window covers the time window during which motor neurons undergo programmed cell death during zebrafish early development. We have added this information to the revised manuscript.

    We frequently used “early development” in this manuscript to describe our observation. However, we missed “early” in our title. We therefore have added this ket word of “early” in the title in the revised manuscript.

    Previous studies in zebrafish have shown that the production of spinal cord motor neurons largely ceases before 48 hpf, and then the motor neurons remain largely constant until adulthood (doi: 10.1016/j.celrep.2015.09.050; 10.1016/j.devcel.2013.04.012; 10.1007/BF00304606; 10.3389/fcell.2021.640414). Our observation time window covers the major motor neuron production process. Therefore, we believe that neurogenesis will not affect our findings and conclusions.

    We discussed the engulfment of dead motor neurons by other types of cells in the discussion section.

    (2) The transgenic line is perhaps the most meaningful contribution to the field as the work stands. However, the mnx1 promoter is well known for its non-specific activation - while the images suggest the authors' line is good, motor neuron markers should be used to validate the line. This is especially important for assessing this population later as mnx1 may be turned off in mature neurons.

    The mnx1 promoter has been widely used to label motor neurons in transgenic zebrafish. Previous studies have shown that most of the cells labeled in the mnx1 transgenic zebrafish are motor neurons. In this study, we observed that the neuronal cells in our sensor zebrafish formed green cell bodies inside of the spinal cord and extended to the muscle region, which is an important morphological feature of the motor neurons.

    Reviewer 2:

    We thank the reviewer for the time and effort in making very useful comments and suggestions for our manuscript.

    The FRET-based programmed cell death biosensor described in this manuscript could be very useful. However, the authors have not considered what is already known about the development and programmed cell death of zebrafish spinal motor neurons, and potential differences between motor neuron populations innervating different types of muscles in different vertebrate models. Without this context, the application of their new biosensor tool does not provide new insights into zebrafish motor neuron programmed cell death. In addition, the authors have not carried out controls to show the efficacy and specificity of their morpholinos. Nor have they described how they counted dying motor neurons, or why they chose the specific developmental time points they addressed. These issues are addressed more specifically below.

    (1) Lines 12-13: Previous studies in zebrafish showed death of identified spinal motor neurons.

    Line 103: In Figure 2A the cell body in the middle is that of identified motor neuron VaP. VaP death has previously been described in several publications. The cell body on the right of the same panel appears to belong to an interneuron whose axon can be seen extending off to the left in one of the rostrocaudal axon bundles that traverse the spinal cord. Higher-resolution imaging would clarify this.

    Lines 163-164: Is this the absolute number of motor neurons that died? How were the counts done? Were all the motor neurons in every segment counted? There are approximately 30 identifiable VaP motor neurons in each embryo and they have previously been reported to die between 24-36 hpf. So this analysis is likely capturing those cells.

    Our study examined the overall motor neuron apoptosis rather than a specific type of motor neuron death, so we did not emphasize the death of VaP motor neurons. We agree that the dead motor neurons observed in our manuscript contain VaP motor neurons. However, there were also other types of dead motor neurons observed in our study. The reasons are as follows: 1) VaP primary motor neurons die before 36 hpf, but our study found motor neuron cells died after 36 hpf and even at 84 hpf (revised Figure 4A). 2) The position of the VaP motor neuron is together with that of the CaP motor neuron, that is, at the caudal region of the motor neuron cluster. Although it’s rare, we did observe the death of motor neurons in the rostral region of the motor neuron cluster (revised Figure 2C). 3) There is only one or zero VaP motor neuron in each motor neuron cluster. Although our data showed that usually one motor neuron died in each motor neuron cluster, we did observe that sometimes more than one motor neuron died in the motor neuron cluster (revised Figure 2C). We included this information in the revised discussion.

    (2) Lines 82-83: It is published that mnx1 is expressed in at least one type of spinal interneuron derived from the same embryonic domain as motor neurons.

    The mnx1 promoter has been widely used to label motor neurons in transgenic zebrafish. Previous studies have shown that most of the cells labeled in the mnx1 transgenic zebrafish are motor neurons. In this study, we observed that the neuronal cells in our sensor zebrafish formed green cell bodies inside of the spinal cord and extended to the muscle region, which is an important morphological feature of the motor neurons.

    Furthermore, a few of those green cell bodies turned into blue apoptotic bodies inside the spinal cord and changed to blue axons in the muscle regions at the same time, which strongly suggests that those apoptotic neurons are not interneurons. Although the mnx1 promoter might have labeled some interneurons, this will not affect our major finding that only a small portion of motor neurons died during zebrafish early development.

    (3) Lines 161-162: Although this may be the major time window of neurogenesis, there are many more motor neurons in adults than in larvae. Neither of these references describes the increase in motor neuron numbers over this particular time span, so the rationale for this choice is unclear.

    Lines 168-171: It is known that later developing motor neurons are still being generated in the spinal cord at this time, suggesting that if there is a period of programmed cell death similar to that described in chick and mouse, it would likely occur later. In addition, most of the chick and mouse studies were performed on limb-innervating motor neurons, rather than the body wall muscle-innervating motor neurons examined here.

    Lines 237-238: Especially since new motor neurons are still being generated at this time.

    Previous studies have shown that the production of spinal cord motor neurons largely ceases before 48 hpf in zebrafish, and then the motor neurons remain largely constant until the adulthood (doi: 10.1016/j.celrep.2015.09.050; 10.1016/j.devcel.2013.04.012; 10.1007/BF00304606; 10.3389/fcell.2021.640414). Our observation time window covers the major motor neuron production process. Therefore, we believe that neurogenesis will not affect our data and conclusions.

    The death of motor neurons in limb-innervating motor neurons has been extensively studied in chicks and rodents, as it is easy to undergo operations such as amputation. However, previous studies have shown this dramatic motor neuron death does not only occur in limb-innervating motor neurons but also occurs in other spinal cord motor neurons (doi: 10.1006/dbio.1999.9413). In our manuscript, we studied the naturally occurring motor neuron death in the whole spinal cord during the early stage of zebrafish development.

    (4) Lines 184-187: Previous publications showed that death of VaP is independent of limitations in muscle innervation area, suggesting it is not coupled to muscle-derived neurotrophic factors.

    Lines 328-334: There have been many publications describing appropriate morpholino controls. The authors need to describe their controls and show that they know that the genes they were targeting were downregulated.

    For the morpholinos, we did not confirm the downregulation of the target genes. These morpholino-related data are a minor part of our manuscript and shall not affect our major findings. We have removed the neurotrophic factors and morpholino-related data in the revised manuscript.

  11. Version published to 10.7554/elife.95691.2 on eLife
  12. Version published to 10.1101/2024.01.26.577434v2 on bioRxiv
  13. eLife

    eLife assessment

    The authors have developed a biosensor for programmed cell-death. They use this biosensor to provide valuable measurements of cell death in a specific early time window of development. However, the title and the discussion suggest a broader window of applicability of the results. The evidence supporting the claims is therefore incomplete. The authors should modify the introduction and discussion to examine their work in the context of extant literature and modify their title to reflect the conclusion that "Zebrafish live imaging reveals around 2%of motor neurons die through apoptosis during a 24-120 hour window in early development".

  14. eLife

    Reviewer #1 (Public Review):

    Summary:

    The authors aim to measure the apoptotic fraction of motorneurons in developing zebrafish spinal cord to assess the extent of neuronal apoptosis during the development of a vertebrate embryo in an in vivo context.

    Strengths:

    The transgenic fish line tg (mnx1:sensor C3) appears to be a good reagent for motorneuron apoptosis studies, while further validation of its motorneuron specificity should be performed.

    Weaknesses:

    The results do not support the conclusions. The main "selling point" as summarized in the title is that the apoptotic rate of zebrafish motorneurons during development is strikingly low (~2% ) as compared to the much higher estimate (~50%) by previous studies in other systems. The results used to support the conclusion are that only a small percentage (under 2%) of apoptotic cells were found over a large population at a variety of stages 24-120hpf. This is fundamentally flawed logic, as a short-time window measure of percentage cannot represent the percentage in the long term. For example, at any year under 1% of the human population dies, but over 100 years >99% of the starting group will have died. To find the real percentage of motorneurons that died, the motorneurons born at different times must be tracked over the long term or the new motorneuron birth rate must be estimated.

    A similar argument can be applied to the macrophage results. Here the authors probably want to discuss well-established mechanisms of apoptotic neuron clearance such as by glia and microglia cells.

    The conclusion regarding the timing of axon and cell body caspase activation and apoptosis timing also has clear issues. The ~minutes measurement is too long as compared to the transport/diffusion timescale between the cell body and the axon, caspase activity could have been activated in the cell body, and either caspase or the cleaved sensor moves to the axon in several seconds. The authors' results are not high-frequency enough to resolve these dynamics

    Many statements suggest oversight of literature, for example, in the abstract "However, there is still no real-time observation showing this dying process in live animals.".

    Many statements should use more scholarly terms and descriptions from the spinal cord or motor neuron, neuromuscular development fields, such as line 87 "their axons converged into one bundle to extend into individual somite, which serves as a functional unit for the development and contraction of muscle cells"

    The transgenic line is perhaps the most meaningful contribution to the field as the work stands. However, the mnx1 promoter is well known for its non-specific activation - while the images suggest the authors' line is good, motor neuron markers should be used to validate the line. This is especially important for assessing this population later as mnx1 may be turned off in mature neurons.

    Overall, this work does not substantiate its biological conclusions and therefore does not advance the field. The transgenic line has the potential to address the questions raised but requires different sets of experiments. The line and the data as reported are useful on their own by providing a short-term rate of apoptosis of the motorneuron population.

  15. eLife

    Reviewer #2 (Public Review):

    Summary:

    Jia and colleagues developed a fluorescence resonance energy transfer (FRET)-based biosensor to study programmed cell death in the zebrafish spinal cord. They applied this tool to study the death of zebrafish spinal motor neurons.

    Strengths:
    Their analysis shows that the tool is a useful biosensor of motor neuron apoptosis in living zebrafish.

    Weaknesses:
    However, they have ignored significant literature describing the death of an identified zebrafish motor neuron, expression of the mnx gene in interneurons that are closely related to motor neurons, the increase in number of zebrafish motor neurons over developmental time, and potential differences between the limb-innervating motor neurons whose death has been characterized in chicks and rodents and the body wall-innervating motor neurons whose death they characterized using their biosensor. Thus, although their new tool is likely to be useful in the future, it does not provide new insights into zebrafish motor neuron programmed cell death.

  16. eLife

    Author response:

    We are grateful to the reviewers for recognizing the importance of our work and for their helpful suggestions. We will revise our manuscript in the revised version. However, we’d like to provide provisional responses now to answer the key questions and comments from the reviewers.

    (1) Both reviewers asked why we chose 24-120 hpf to measure the apoptotic rates. We chose this time window based on the following two reasons: 1) Previous studies showed that although the motor neuron death time windows vary in chick (E5-E10), mouse (E11.5-E15.5), rat (E15-E18) and human (11-25 weeks of gestation), the common feature of these time windows is that they are all the developmental periods when motor neurons contact with muscle cells. The contact between zebrafish motor neurons and muscle cells occurs before 72 hpf, which is included in our observation time window. 2) Zebrafish complete hatching during 48-72 hpf, and most organs form before 72 hpf. More importantly, zebrafish start swimming around 72 hpf, indicating that motor neurons are fully functional.

    Thus, we are confident that this 24-120 hpf time window covers the time window during which motor neurons undergo programmed cell death during zebrafish early development. We frequently used “early development” in this manuscript to describe our observation. However, we missed “early” in our title. We will add “early” in the title in the revised version.

    (2) Both reviewers also asked about the neurogenesis of motor neurons. Previous studies have shown that the production of spinal cord motor neurons largely ceases before 48 hpf and then the motor neurons remain largely constant until adulthood. Our observation time window covers the major motor neuron production process. Therefore, we believe that neurogenesis will not affect our data and conclusions.

    (3) Both reviewers questioned the specificity of using the mnx1 promoter to label motor neurons. The mnx1 promoter has been widely used to label motor neurons in transgenic zebrafish. Previous studies have shown that most of the cells labeled in the mnx1 transgenic zebrafish are motor neurons. In this study, we observed that the neuronal cells in our sensor zebrafish formed green cell bodies inside of the spinal cord and extended to the muscle region, which is an important morphological feature of the motor neurons. Furthermore, a few of those green cell bodies turned into blue apoptotic bodies inside the spinal cord and changed to blue axons in the muscle regions at the same time, which strongly suggests that those apoptotic neurons are not interneurons. Although the mnx1 promoter might have labeled some interneurons, this will not affect our major finding that only a small portion of motor neurons died during zebrafish early development.

    (4) Reviewer 2 is concerned that the estimated 50% of motor neuron death was in limb-innervating motor neurons but not in body wall-innervating motor neurons. The death of motor neurons in limb-innervating motor neurons has been extensively studied in chicks and rodents, as it is easy to undergo operations such as amputation. However, previous studies have shown this dramatic motor neuron death does not only occur in limb-innervating motor neurons but also occurs in other spinal cord motor neurons. In our manuscript, we studied the naturally occurring motor neuron death in the whole spinal cord during the early stage of zebrafish development.

    (5) Reviewer 2 mentioned that we ignored the death of an identified motor neuron. Our study was to examine the overall motor neuron apoptosis rather than a specific type of motor neuron death, so we did not emphasize the death of VaP motor neurons. We agree that the dead motor neurons observed in our manuscript contain VaP motor neurons. However, there were also other types of dead motor neurons observed in our study. The reasons are as follows: 1) VaP primary motor neurons die before 36 hpf, but our study found motor neuron cells died after 36 hpf and even at 84 hpf. 2) The position of the VaP motor neuron is together with that of the CaP motor neuron, that is, at the caudal region of the motor neuron cluster. Although it’s rare, we did observe the death of motor neurons in the rostral region of the motor neuron cluster. 3) There is only one or zero VaP motor neuron in each hemisegment. Although our data showed that usually one motor neuron died in each hemisegment, we did observe that sometimes more than one motor neuron died in the motor neuron cluster. We will include this information in the revised manuscript.

    (6) For the morpholinos, we did not confirm the downregulation of the target genes. These morpholino-related data are a minor part of our manuscript and shall not affect our major findings. Thus, we didn’t think we missed “important” controls. We will perform experiments to confirm the efficiency of the morpholinos or remove these morpholino-related data from the revised version.

  17. Version published to 10.7554/elife.95691.1 on eLife
  18. Version published to 10.1101/2024.01.26.577434v1 on bioRxiv